The long range goal of this research is to understand the molecular basis of the process by which Rhizobia nodulate legumes. In the proposal we focus on genes involved in nodulation that are present in Rhizobium meliloti. We will develop methodologies for the study of plasmids in R meliloti including: expanding the number of transposon systems useful for insertion mutagenesis, developing a convenient system for cloning in R. meliloti, and developing a "maxicell" system. A set of nodulation (nod) defective mutants will be generated by insertion mutagenesis. We will prepare restriction-endonuclease maps of the portions of R. meliloti plasmids carrying nod genes. The nod genes will be cloned and the relevant regions of DNA identified by subcloning and insertion mutagenesis. The non gene products will be identified by examination of the nod mutants for changes in rhizobial surface components and use of maxicells to visualize plasmid-coded proteins.